The present invention relates to a process for the preparation of a system having good controlled release behavior, and to microspheres with a good controlled release behavior. More in particular the invention relates to a method for the preparation of microencapsulated colloidal systems, i.e., microspheres which comprise colloidal systems, such as liposomes. These microencapsulated colloidal systems can be used as controlled release systems for the delivery of active ingredients in in vivo and in vitro applications.
The fast developments in the biotechnological field lead to a large number of pharmaceutically interesting products, esp. proteins, peptides and genes. Such products can suitably be used in the treatment of life-threatening diseases, e.g. cancer, and several types of viral, bacterial and parasitic diseases.
Due to their nature, proteins and proteinaceous products, e.g. peptides, which group of products will be referred to as protein drugs herein-below, cannot efficiently be administered orally. They have to be brought in the system parenterally, i.e. by injection. The pharmacokinetic profile of these products is such that injection of the product per se requires a frequent administration. In other words, since protein drugs are chemically and physically unstable in the gastro intestinal tract and generally have a short active residence time in the human or animal body, multiple injections in a short time are required to attain a therapeutic effect. It will be evident that this is inconvenient for patients requiring these protein drugs.
For this reason, there is a need for delivery systems which have the capacity for sustained release. A number of options for such systems have been proposed in the art, such as the use of synthetic biodegradable, rather well-defined polymers to control the release of encapsulated drugs.
One of the options described in the prior art is the use of microspheres and nanospheres made of polymeric materials. These microspheres or nanospheres are spherical particles, spherical capsules, nanocapsules or nanoparticles having a particle diameter between about 0.1 xcexcm and about 100 xcexcm. In this description and the claims, the reference to microspheres also encompasses microparticles, microcapsules, nanospheres, nanoparticles and nanocapsules. Widely used polymers to prepare these microspheres are poly lactic acid and copolymers of lactic acid and glycolic acid. The polymers should preferably be biodegradable to avoid removal of the polymer carrier after use.
The hitherto known preparation methods for drug containing controlled or sustained release systems generally involve the use of organic solvents. Organic solvents may lead to structural changes in protein structure, esp. in the secondary and tertiary structure. Such changes may lead to a denaturation of the protein drug. Since these structural changes normally lead to a loss in pharmacological activity and the occurrence of undesired side-effects, such changes are undesirable, as will be apparent. Moreover, the use of organic solvents is not desirable from an environmental point of view, either.
Further, it is hardly possible to avoid that traces of organic solvents will remain in or on the microspheres produced. Especially, when toxic solvents are used, such as the widely applied solvents chloroform and dichloromethane, this is a problem.
Another problem is that it is difficult to encapsulate proteins in polymeric matrices in a reproducible way. It is of the utmost importance that predictable and reproducible amounts of proteins or other encapsulated products to be used as drugs are released.
Polymeric hydrogels, i.e., polymeric networks that contain a considerable amount of water, are also widely studied as controlled release systems. Although polymeric hydrogels can be applied successfully as controlled release systems, there remains a need to further modify the release profiles obtained. In particular the lag time, i.e., the time after which the onset of release occurs, and the duration of the pulse in case of pulsed release are parameters that determine the success of the application of a controlled release system to a large extent.
One of the hydrogel systems that has been used in the preparation of delivery systems for protein drugs comprises crosslinked dextrans obtained by radical polymerization of methacrylate derivatized dextran (dex-MA). In this respect, reference is made to van Dijk-Wolthuis et al. in Macromolecules 28, (1995), 6317-6322 and to Van Dijk-Wolthuis et al. in Macromolecules 30, (1997), 3411-3413.
It appeared that the release of proteins from these hydrogels depends on and can be controlled by the degree of crosslinking and the initial water content of the gel (Hennink et al., J. of. Contr. Rel. 39 (1996), 47-57, the contents thereof being incorporated herein by reference).
Encompassed drugs are released from these hydrogels or polymeric microspheres during biodegradation of the polymeric material and/or by diffusion.
Drugs are usually loaded into hydrogels or microspheres derived hereof either by equilibration in a drug-containing solution followed by drying (see e.g. Kim et al. in Pharm. Res. 9(3) (1992) 283-290) or by incorporation of the drug during the preparation of the hydrogel or microspheres (see e.g. Heller et al. in Biomaterials 4 (1983) 262-266). Both techniques have a number of disadvantages other than those arising from any organic solvents used.
Loading by equilibration normally leads to a rather low drug content in the delivery system due to entropic exclusion: larger molecules enter the hydrogel with more difficulty than smaller ones. This is especially the case, when the drug is a macromolecular compound. Unless the pore size of the hydrogel or the microsphere is rather large, the macromolecules will only adsorb onto the outer surface, which may, after application, lead to a burst release in the human or animal system or in vitro. Further, the solvent phase containing the drug, which phase is contacted with the delivery system to load the delivery system, has to be removed from the hydrogel or the microspheres. This can produce the migration of the drug to the surface of the delivery system, and, hence, to a non-homogeneous drug distribution. This tends to result in a significant burst release of the drug, as well, which generally is not desired.
A suitable loading process for incorporating macromolecular drugs is aimed at.
In an article in Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 22 (1995), 145-146, Gehrke et al. have described a technique wherein loading levels higher than obtainable by solution sorption, hence, higher than about 0.1 wt. %, can be achieved in purified, pre-formed hydrogels. The loading technique is based on the fact that certain polymer mixtures split into separate phases when dissolved in water. Proteins dissolved in such a system distribute unevenly between the phases. This principle also holds when one of the polymer phases is a crosslinked gel.
In particular, Gehrke et al. describe a crosslinked dextran gel/poly(ethylene glycol) system, and a crosslinked hydroxypropylcellulose gel/poly(vinyl alcohol) system. Proteins present in an aqueous solution containing beads of the gel are, after the addition of the non-crosslinked second polymer, adsorbed on the beads and partly absorbed through meshes or pores in the bead surfaces.
A disadvantage of this technique is that the proteinaceous material is to a major extent only adsorbed to the beads, which means that if the phase containing the second polymer is replaced by another aqueous system a fast removal of the proteins from the beads is observed. Only when large amounts of pores having a diameter larger than the size of the proteinaceous material to be loaded are present in the bead surfaces, some absorption may occur. This adsorption and limited absorption behavior has an undesirable effect on the release of the proteinaceous material from the beads.
To additionally illustrate the undesired release behavior, it is noted that the profiles shown in the article arexe2x80x94from a pharmacological point of viewxe2x80x94entirely unsuitable to be used in controlled release systems.
In EP-A-0 213 303 a method for producing spherical polymer particles from systems containing two liquid aqueous phases is described. One of the two phases is dispersed in the form of droplets in the other phase to form an emulsion. Subsequently, the droplets are caused to solidify. In the phase to be dispersed, a macromolecular substance may be dissolved. Further, low molecular substances such as medicaments, vaccines and insecticides can be chemically bonded to the particle forming substance in the dispersed phase. Nothing is being said about the release behavior of the dissolved substance, nor over the application of the spherical polymer particles formed or the size thereof.
The principle of affinity partitioning in PEG-containing two-phase systems is also known from Gxc3x6te Johansson, Affinity Partitioning in PEG-containing Two-phase Systems In: Topics in Applied Chemistry; Poly(ethylene glycol) chemistry, Biotechnological and Biomedical Applications, Ed. J. M. Harris, Plenum Press (1992). In this article, a two-phase system is described, which is created when an aqueous solution of dextran and polyethylene glycol (PEG) are mixed. A PEG enriched and a dextran enriched phase are formed. Proteins are partitioned unequally in such systems. These known systems are used in the purification of proteins.
Liposomes have been under investigation as controlled release systems for many years. Liposomes consist of concentric closed membranes, such as a bilayer formed by water-insoluble polar lipids, such as phospholipids. Other substances, such as cholesterol, may also be included in the membrane. These liposomes may vary in diameter from a few tens of nanometers up to micrometers. Both polar and non-polar drugs or enzymes can be encapsulated as active ingredient within liposomes. Proteins can be encapsulated in liposomes very well. When using liposomes as controlled release systems, initial high concentrations of proteins in the blood may be prevented. Such high concentrations are often undesirable, because they may lead to adverse effects in the patient and to degradation of the protein. The release of the active ingredient from the liposome may be triggered by the destabilization of the bilayer.
(Micro)encapsulation of liposomes has been reported in several instances. For example, Kibat et al. (FASEB J., 4 (1990) 2533-2539) describe microcapsules composed of a hydrogel matrix of calcium alginate. These microcapsules contain liposomes. Pulsatile release of compounds from the liposomes is obtained by coating the liposomes with phospholipase A2 enzyme.
Cohen et al. (Proc. Natl. Acad. Sci. USA, 88 (1991) 10440-10444) and Cohen et al. (Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 16 (1989) 71-72) also describe alginate-microencapsulated liposomes. These microencapsulated liposomes are characterized by a release profile which starts immediately, i.e., show no lag time. In addition, the release is gradual rather than pulsed.
U.S. Pat. No. 4,921,757 discloses liposomes that are encapsulated in a solid permeable plate of e.g. agarose or polyacrylamide, or in microparticles based on crosslinked alginate with a polykationogenic skin.
Yeung et al. (J. Microencapsulation, 5 (1988) 331-337) describe liposomes incorporated in nylon based microcapsules, which are prepared using a interfacial polycondensation process. The release profiles of these microencapsulated liposomes shows no lag-time. Moreover, the use of nylon makes in vivo application unlikely.
Bochot et al. (International Journal of Pharmaceutics, 162 (1998) 119-127) describe an ocular delivery system based on liposomes dispersed in a thermosensitive polyoxyethylene-polyoxypropylene copolymer gel. The system is prepared by adding liposomes to the gel.
Weiner et al. (Journal of Pharmaceutical Sciences, 74 (1985) 922-925) describe the use of a collagen gel for encapsulating liposomes. After the liposomes have been added to the collagen, gel formation is initiated by changing the temperature or the pH.
Alamelu et al. (Carbohydrate Polymers, 24 (1994) 215-221) describe a delivery system based on sequestered liposomes coupled to a chitosan gel. Gel formation is effected by addition of an alkaline solution.
Finally, DiTizio et al. (Biomaterials, 19 (1998) 1877-1884) describe a liposomal hydrogel. The gel is formed by changing the pH.
All of these prior art microencapsulated liposomes rely on enzymes to effect the release of compounds from the liposomes or do not show desirable release characteristics. In addition, the encapsulation efficiency of the prior art methods is usually too low.
The present invention is aimed at providing a new injectable, patient friendly delivery system for active ingredients, such as protein drugs or other drugs, which system is safe and biodegradable, and which system possesses well controllable delivery kinetics. The period wherein drug delivery should be guaranteed depends on the active ingredient used that preferably is protein drug, and varies between a number of days up to more than one year. In addition, high degrees of loading in the delivery system should be obtained. Moreover, the system of the present invention should be produced without needing the use of organic solvents.
It is another objective of the present invention to provide a method for the preparation of microencapsulated colloidal systems, such as liposomes, which colloidal systems may be associated with an active ingredient, which microencapsulated colloidal systems display a desirable release profile, i.e., a high release of active ingredient (preferably more than 85%), a pulsed release profile having a suitable lag-time, while these release profiles can be controlled by the method of preparation. The desirable release profile should also hold for the active ingredient which may be associated with the colloidal systems. The definition of a suitable lag-time depends on the type of application. When vaccines are used as an active ingredient, a suitable lag-time is between two weeks to one year, preferably 1-3 months.
It is another objective of the present invention to provide such a method which enables a high encapsulation efficiency of the colloidal systems, i.e., an encapsulation efficiency of more than 80%, preferably more than 90%.
The present invention is directed to a combination of colloidal systems and hydrogels used for controlled release of active ingredients.
Suitable colloidal systems which may be used in the present invention are liposomes; iscoms; polyplexes, i.e. combinations of (cationic) polymers and DNA; lipoplexes, i.e. combinations of (cationic) lipids and DNA; nanoparticles, i.e. polymer based spheres in the nanometer size range; solid lipid particles in the colloidal size range (see for example R. H. Muller et al., Pharm. Research 14 (1997) 458-462); emulsions, such as intralipid-like systems; any other entity in the colloidal size range having a low water solubility; and combinations thereof.
The combinations of colloidal systems and hydrogels according to the present invention will be illustrated hereinafter using liposomes as the colloidal system. It is to be understood that were liposomes are mentioned hereinafter, these liposomes can be replaced by any of the suitable colloidal systems mentioned above, without departing from the spirit of the invention.
The problems mentioned above are solved by a specific preparation method of controlled release systems, such as microspheres, wherein water is used as the solvent. The use of water as sole solvent system is advantageous from an environmental point of view, because of toxicological considerations and, especially, because of reasons of protein stability.
In a first aspect, the present invention provides a method for the preparation of a controlled release system, comprising:
(a) forming an aqueous two-phase system from two water soluble polymers and water and at least one releasable entity, such as a liposome or another colloidal system, the two water soluble polymers being incompatible in solution, at least one of these polymers being crosslinkable, the crosslinkable polymer phase being emulsified in the other polymer phase; and the at least one releasable entity being soluble or dispersible in the crosslinkable polymer phase in the aqueous solution;
(b) allowing the releasable entity to distribute in the crosslinkable polymer phase; and
(c) crosslinking of the crosslinkable polymer.
In a preferred embodiment, the crosslinking is carried out to such a degree that the pores (meshes) in the crosslinked structure eventually formed are substantially smaller than the size of the releasable entity.
By emulsifying an aqueous crosslinkable polymer in a continuous phase comprised of water and a polymer which is not compatible with the crosslinkable polymer, and crosslinking the discontinuous phase, particles are formed. The particle size of the crosslinked polymer particles can be adjusted by changing the viscosity of one or both phases, for example by choosing polymers of different molecular weight, or by changing the volume ratio of both phases (see e.g. Stenekes et al., Pharm. Res. 15 (1998) pp. 557-561, the contents of this document being incorporated herein by reference). In this way a narrow particle size distribution can be obtained, as is described herein-below in more detail.
In a further aspect, the present invention is directed to microspheres, at least 80 wt. % thereof having a particle size of between 100 nanometer and 100 xcexcm, which microspheres are comprised of a degradable, crosslinked polymer encapsulating at least one releasable entity, such as a liposome, the pore size of the crosslinked polymer being equal or preferably smaller than the size of the releasable entity. These microspheres are obtainable by using the process of the invention, and are free from organic solvents. Dependent on the application of the microspheres, the size can e.g. be adjusted between 1 and 50 xcexcm, preferably between 2 xcexcm and 25 xcexcm, such as between 5 and 15 xcexcm.
When the pore sizes or meshes of the crosslinked polymer are equal or smaller than the hydrodynamic diameter size of the releasable component, the releasable component is essentially released when the polymer is degraded. More in particular, in this embodiment, the crosslinked structure must be degradable in the human or animal body, so that the encapsulated releasable entity can leave the crosslinked matrix. If on the other hand, the pore sizes or meshes of the crosslinked polymer are larger than the size of the releasable component, the releasable component is at least partially released by diffusion. The pore size of the crosslinked product obtained by the process of the present invention in this way provides a perfect tool to control the release. Further, the efficiency of the incorporation of a releasable entity in such a polymer structure is very high, while the degree of loading can be adjusted up to the saturation concentration of the entity to be released.
The degradability of the crosslinked structure can be regulated in a number of ways. As a first example, it is noted that bonds can be incorporated, which are hydrolysable under physiological conditions. In this respect, reference can be made to the European patent application 96201821.4 and WO-A-98/00170, the contents of which documents are incorporated herein by reference. These patent applications teach hydrogels comprising hydrolytically labile spacers between different polymer chains. The hydrolytically labile spacers described therein can be suitably used in the present invention and the above mentioned patent applications are incorporated herein by reference.
Another example to control the degradability is the coencapsulation of an enzyme or chemical substance capable of breaking bonds in the crosslinked polymer. In a preferred embodiment of the process of the present invention the crosslinkable polymer is a dextran polymer. In this embodiment a dextranase can be added to the aqueous two-phase system before the crosslinking step or added afterwards.
The dextran polymer can suitably be used in the process of the present invention together with Pluronic(copyright) or a polyethylene glycol, which latter polymer is preferred to be used in the process of the present invention.
The product aimed at by the process of the present invention can be separated from the other polymer phase using conventional techniques, for instance centrifugation and decantation.
In a first step of the process of the present invention an aqueous two-phase system is formed. This two-phase system comprises water, and at least two water soluble polymers, which polymers are incompatible in solution. Preferably an entity to be released is also present, although it is possible to add the entity to be released after the crosslinking step. The colloidal systems used in the present invention are however generally too large to enter the microspheres after the crosslinking step in a sufficient amount, in particular when liposomes are used as the colloidal system. Therefore it is preferred to add these colloidal systems before the crosslinking step. At least one of the polymers present in the aqueous phase is chemically or physically crosslinkable, and the crosslinkable polymer phase is emulsified in the other aqueous polymer phase.
The polymers used can be chosen dependent on the nature of the entity to be released. It is preferred that the entity to be released will have a clear preference for the crosslinkable polymer phase. In that case, the highest possible degree of loading, theoretically up to the saturation concentration, in the microspheres to be made, can be obtained.
It is not critical which crosslinkable polymer is used. However, if the controlled release system comprising the polymer in crosslinked form is intended to be brought into a human or animal body, the polymer should be pharmaceutically acceptable and preferably should be degradable. Suitable crosslinkable water soluble polymers are dextrans and derivatized dextrans, starches and starch derivatives, cellulose derivatives such as hydroxyethyl and hydroxypropyl cellulose, polyvinylpyrrolidone, proteins and derivatized proteins, and so on. The molecular weight of the crosslinkable polymers used normally lies between 1,000 and 1,000,000 Da. It is noted that with a higher molecular weight of the polymer, a better phase separation is generally obtained in the aqueous solution used in the process of the invention.
The person skilled in the art will have the knowledge to choose the crosslinkable polymer and the crosslinking conditions required for the emulsion prepared. For instance, dextrans can be crosslinked with methylacrylate or methacrylate groups. Another example is a system comprising PVP as the external phase and dextran as the emulsified phase, wherein the dextran is crosslinked through the presence of isocyanates.
Further, reference is made to crosslinking using radiation. Dex-MA (methacrylate derivatized dextran) can e.g. be polymerized using small dosages of xcex3-radiation, such as less than 0.1 Mrad. An advantage of this embodiment is that in one step sterile microparticles can be obtained. Further, crosslinking by UV radiation and physical crosslinking using e.g. hydrophobic tails coupled to a polymer are possible techniques.
In a preferred embodiment, the crosslinkable polymer is a temperature sensitive polymer such as poly-N-isopropyl-acrylamide, which polymer can, e.g., be present as a graft on another polymer such as a dextran. Hydrogels of these polymers show increasing swelling behavior at degreasing temperatures. This makes it possible that releasable material can easily penetrate in the hydrogel after the crosslinking reaction. By subsequently raising the temperature, e.g. to a value of 37xc2x0 C., the meshes in the hydrogel shrink, thereby capturing the releasable entity.
The polymer which is present in the aqueous continuous phase can be any polymer which is incompatible with the crosslinkable polymer. Although this polymer may also be crosslinkable, but of course not under the reaction conditions used for the crosslinking of the discontinuous polymer phase, this is not preferred. Examples of suitable polymers incompatible with the polymer to be crosslinked are poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) (in combination with e.g. dextrans and dextran derivatives, starches and starch derivatives, PVP, and water soluble cellulose derivatives).
The release of the releasable entity depends on a number of variables, which can be used to tailor the delivery as desired. One of these variables is the size of the microspheres. The size can be adjusted by carefully modifying the process circumstances and formulation parameters in the emulsifying step. For instance, the water content, the presence of hydrophobic groups on any one of the polymers or mixtures of polymers used, the viscosity of the continuous and discontinuous phase, and the electrical charge on the at least two polymers used are examples of tools to adjust the size of the microspheres or microparticles to be produced. In addition, emulsifiers can be added. Suitable emulsifiers are copolymers, preferably block-copolymers, of units of the two incompatible polymers, e.g. a block-copolymer of PEG and dextran, used to create the two-phase system.
To further guarantee a controlled release, the crosslinked polymer should preferably be degradable.
As said herein-above, it is important that the two water soluble polymers are incompatible with one another, so that a two-phase system is obtained after the two polymers have been added to each other in an aqueous solution. Whether or not a two-phase system will be obtained depends not only on the nature of the two polymers involved, but also on the conditions under which they are added. Factors that are relevant in this regard are the molecular weight of the polymers, their concentrations in the aqueous solution, the temperature at which they are added to one another, and so forth. It is part of the standard skills of the artisan to determine a phase diagram for any combination of polymers that can be used, and thus to choose suitable conditions for obtaining a phase separation.
In the attached FIG. 8 a phase diagram of a water/PEG/dextran ternary system is shown as example. When the starting-composition is below the binodal (- - -), a one phase system is present, whereas above the binodal, two coexisting phases are formed: one enriched in polymer 1 (composition x1) and the other enriched in polymer 2 (composition x2)xe2x80x94x1 and x2 are connected via a tie-line (_). All systems prepared using starting-compositions on the same tie-line separate into phases of constant composition. For a given starting-composition, the volume ratio of the coexisting phases x1/x2 equals y2/y1.
As indicated herein-above, the releasable entity can be a protein drug. However, it is also possible to encapsulate a pharmacon or antigen or other active agents containing colloidal systems, such as nanoparticles or microparticles, e.g. liposomes and iscoms. The encapsulation of this type of particles has the advantage of preventing the occurrence of a too fast release of the encapsulated entity, or, said in other words, burst-effects can be avoided in a more secure way.
The microencapsulated colloidal systems obtained by the method of the invention comprise a matrix of a degradable polymer in which the colloidal systems, such as liposomes, are present. Active ingredients may be present in the colloidal systems. It is a considerable advantage of the present invention that these active ingredients may be chosen in principle regardless their compatibility with the polymeric matrix, i.e. it is not required that the active ingredients are soluble or dispersible in the matrix itself, since it is sufficient that these active ingredients are compatible with the colloidal system. This enables the preparation of release systems comprising active ingredients which could only with difficulty or only with additional measures, be incorporated in release systems of the prior art.
The method of the invention thus enables microspheres for e.g. the delayed or pulsed release of all types of active ingredients. When liposomes are used as the colloidal systems, the presence of the bimolecular lipid membrane layer enables the incorporation of hydrophobic substances, which could not be brought in conventional hydrogel systems. These hydrophobic substances can be incorporated in, or associated with the lipid bilayer.
Examples of colloidal particles to be encapsulated are: iscoms, lipoplexes, polyplexes, nanoparticles and solid liponanoparticles.
Iscoms may be released by the microspheres slowly or in a pulsed manner. This allows the designer of vaccines to manipulate the immune response induced by the antigen-carrying iscoms. E.g., if a booster is desired, the microspheres can induce a booster effect by pulsed release of the iscoms after a predetermined delay time.
It may be desired to have access to delivery systems that release polyplexes and lipoplexes (complexes of genetic material and condensing polymer (mixtures) or (mixtures) in a time controlled way. The microspheres are able to do that.
The same applies to nanoparticles and lipospheres, which can be loaded with therapeutically active material or antigens. Nanoparticles are colloidal particles based on e.g. polycyanoacrylates. Solid lipid nanoparticles are colloidal particles based on lipids which are in the solid phase at body temperature. Nanoparticles and solid lipid nanoparticles can be released from the microspheres in similar patterns as liposomes. Drug release from these colloidal particles depends on their building components and manufacturing conditions.
Moreover, by incorporating the active ingredient in the colloidal system, the active ingredient is protected from the reactions that take place during the subsequent polymerization step, which reactions could cause undesired oxidation of the active ingredient.
With the method of the invention, it is possible to provide microcapsules which comprise 10 wt. % or more of the colloidal system. Usually, the amount of incorporated colloidal system will be from about 5-10 wt. % but this amount will vary with the specific conditions used.
The amount of active ingredient which may in total be incorporated as well in the microsphere, as the efficiency of this incorporation, will also vary strongly depending on synthesis conditions and envisaged application. For example, if a membrane protein is to be incorporated in a liposome, it will be readily absorbed in the bilayer of the liposomes. These liposomes can be incorporated into microspheres with high efficiencies, as was stated above. On the other hand, a hydrophilic protein will be incorporated less efficiently in the liposome, resulting in lower protein content of the final microsphere. If microspheres with a high content of active ingredient are desired, a more hydrophilic colloidal system can be used.
Typically, the amount of active ingredient present in the microspheres prepared with the method of the present invention, will be from about 0.5-50 xcexcg active ingredient/mg microsphere material.
The partition of the entity to be released is primarily determined by the nature of the polymers present in the aqueous two-phase system. This partition can be influenced, e.g. by adding salt to the aqueous system, or by adjusting the pH.
If the releasable entities, such as proteins, are present during the crosslinking step, care should be taken that the integrity of the releasable entities is secured. It should for instance be avoided that proteinaceous material is oxidized by initiator systems etc. In this light, it is noted that adverse effects can be avoided or minimalized by minimalizing the amount of initiator, reducing the polymerization time or adding suitable antioxidantia, such as xcex1-tocopherol.
The separation of the crosslinked structures enclosing the releasable entity from the other phase can be carried out in any conventional way. Preferably, the separation is effected by filtration or centrifugation. The crosslinked structures can subsequently be washed with water and dried. The drying step determines that a pharmaceutically acceptable product can be obtained, having a maintenance term of more than 2 years. A very preferred drying method is spray-drying, although the drying can also be suitably carried out using lyophilization.